Cerebral antioxidants are neuroprotective agents that prevent oxidative injury during normal aerobic metabolism. Ascorbic acid (ascorbate) and glutathione (GSH) are the most abundant antioxidants of low molecular weight in the CNS. At the onset of ischemic injury, energy-dependent mechanisms that maintain cellular levels of ascorbate and GSH fail, so that they and other small molecules are lost brain cells. The underlying premise of this proposal is that loss of intracellular ascorbate and GSH during ischemia leaves brain cells vulnerable to oxidative damage. Despite the central role of ascorbate and GSH in the antioxidant network, surprising little is known about the mechanisms that regulate their homeostasis in the CNS. The long-term objective of the studies is to determine the mechanisms that regulate antioxidant homeostasis in the CNS, which is a necessary first step in the development of appropriate therapies. Preliminary data indicate ascorbate and GSH levels decrease with age and vary with gender. These data are consistent with human epidemiology, which indicate that stroke incidence and severity increase with age and are greater in males than in females. Proposed studies, therefore, will necessarily address factors that govern age and sex differences. Experiments will use HPLC to determine total tissue ascorbate and GSH, and histological methods to evaluate tissue damage and antioxidant neuroprotection. In addition, tools are available to monitor the extracellular levels of ascorbate ([Asc]o). Using voltammetry with carbon fiber microelectrodes, we will detect [Asc]o in brain slice in vitro and after middle cerebral artery occlusion (MCAO) in vivo, then combine these data with total tissue ascorbate content to calculate [Asc]i. Specific aims are: 1. To determine the differential localization of ascorbate and GSH in the CNS, with emphasis on the hypothesis that ascorbate is in neurons and GSH is in glia. We will confirm antioxidant loss in the aging brain and extend gender studies to include antioxidant enzymes and iron. II. To evaluate the mechanism of intra-and extracellular ascorbate homeostasis. In vitro experiments will test the hypothesis that intra- and extracellular ascorbate concentrations ([Asc]i and [Asc]o) are regulated homeostatically and that the dynamics of this process change with age. III. To evaluate where and how ascorbate and GSH protect. In vitro experiments will test the hypotheses that ascorbate and GSH act intracellularly. IV. To determine mechanisms of ascorbate and GSH loss in ischemia. We will extend preliminary data indicating that loss of antioxidants during ischemia is greater in male rats than in females. V. To determine the relationship between loss of intracellular ascorbate and in focal ischemia. Experiments will test the hypothesis that regions with the greatest loss of [Asc]i during ischemia have the greatest extent of damage.